Abstract
During the summer, pregnant ewes experience heat stress, leading to the
occurrence of IUGR lambs. This study aims to explore the biomarkers of
exosomal miRNAs derived from umbilical plasma in both IUGR and normal
Hu lambs. We establish a heat-stressed Hu sheep model during mid-late
gestation and selected IUGR and normal lambs for analysis. Exosomes
from umbilical plasma were separated and small RNA sequencing is used
to identify differentially expressed miRNAs. Next, we utilize MiRanda
to predict the target genes of the differentially expressed miRNAs. To
further understand the biological significance of these miRNAs, we
conduct GO and KEGG pathway enrichment analysis for their target genes.
The study’s findings indicate that oar-miR-411a-5p is significantly
downregulated in exosomes derived from umbilical plasma of IUGR lambs,
while oar-miR-200c is significantly upregulated in the HS-IUGR group
(P < 0.05). Furthermore, GO and KEGG enrichment analysis demonstrate
that the target genes are involved in the Wnt, TGF-beta, and Rap1
signaling pathways. miRNAs found in exosomes have the potential to be
utilized as biomarkers for both the diagnosis and treatment of IUGR
fetuses.
Subject terms: Intrauterine growth, Diagnostic markers
__________________________________________________________________
Small RNA profiling of exosomes derived from umbilical plasma from
lambs exposed to intrauterine growth restriction.
Introduction
Heat stress has a significant impact on both human and animal
production, including growth, development, and reproduction^[32]1.
There are many factors that cause intrauterine growth retardation
(IUGR), such as maternal malnutrition, placental dysfunction, and fetal
factors, genetic factors also account for a major proportion of the
occurrence of IUGR^[33]2,[34]3. In particular, sheep are vulnerable to
heat stress when the temperature rises above 25 °C, which challenges
their natural temperature regulation mechanisms and can lead to reduced
performance and health issues^[35]4. As global warming continues, sheep
will face even more intense and prolonged heat stress^[36]5. As a
result, heat stress is a major factor that affects the development of
the sheep industry. Exposure of sheep to heat stress has increasingly
severe consequences due to the rising temperatures associated with
global climate change^[37]6. When pregnant sheep are exposed to heat
stress, it negatively impacts fetal growth and the weight of newborn
lambs^[38]6. Heat stress begins to affect the fetus during
mid-gestation, and by late gestation, the size of the fetus is less
than two standard deviations from that of a normal sheep^[39]7. During
late gestation and in newborn lambs from heat-stressed ewes, there is a
change in fetal morphology that the average fetal weight and liver
weight decrease, which suggests the presence of IUGR^[40]8.
This IUGR is caused by the elevated ambient temperature experienced by
the ewes during the pregnancy period, and there is a strong correlation
between the maximum temperature of the uterus during pregnancy and the
birth weight of the offspring^[41]9,[42]10. IUGR is a condition where
the weight of the fetus is less than two standard deviations of the
average weight for the same age, or below the 10th percentile of the
normal weight for the same age, and IUGR is one of the significant
complications that can arise during pregnancy^[43]11. Lambs born under
severe natural conditions, such as cold and heat stress, experience a
decrease in birth weight^[44]12. The impact of ambient temperature on
pregnancy varies depending on the stage of gestation, with late
pregnancy being more vulnerable than early pregnancy, and there is an
almost linear correlation between the ewe’s thermal peak and the birth
weight of their offspring^[45]10.
Exosomes, which are membranous vesicles with a diameter of 30–150 nm,
are produced and secreted by living cells^[46]13. They have a round or
cup-like shape that can be observed through electron microscopy^[47]14.
Exosomes are small vesicles that play a crucial role in intercellular
communication, and there are around 10^14 exosomes in the human body,
with each cell producing an average of 1000–10,000 exosomes^[48]15.
These vesicles can be found in almost all tissues, intercellular
spaces, and body fluids, such as blood, saliva, urine, and breast milk,
and they carry various molecules, including proteins, miRNAs, lncRNAs,
circRNAs, and mRNA, which are involved in intracellular signal
transduction and help regulate the biological processes of
cells^[49]16. Exosomes can also be produced by the placenta and have
the potential to serve as biomarkers for pregnancy diagnosis by
carrying miRNA into the maternal circulation^[50]17. In addition,
exosomes secreted by the endometrium enhance the embryo’s adhesion
ability^[51]18. As gestation progresses, the concentration of serum
exosomes from the gestational day of 90 pregnant sheep also
increases^[52]19. Therefore, exosomes play a critical role in both
pregnancy diagnosis and embryo implantation.
To establish a model of heat stress during mid-late gestation, Hu sheep
were used as experimental animals in this study. After the gestation
period, IUGR and healthy lambs were distinguished. Then exosomes from
the umbilical plasma of both IUGR and healthy lambs were separated.
Small RNA sequencing was conducted to identify the small RNAs expressed
in exosomes and to understand their roles in the development of IUGR
due to heat stress. Our findings demonstrated that miRNAs present in
exosomes from umbilical plasma have the potential to serve as
biomarkers for both the diagnosis and treatment of IUGR in fetuses.
Results
Establishment of the heat-stressed Hu sheep model during mid-late gestation
The ambient temperature of the sheep shed fluctuated between 5 and
25 °C in Jan, Feb, and Mar, and undulated from 20 to 40 °C in Jun, Jul,
and Aug (Fig. [53]1a). The effective temperature of pregnant Hu sheep
was similar to the ambient temperature (Fig. [54]1b). The
temperature-humidity index (THI) showed most pregnant Hu sheep were in
a no-heat-stressed state in Jan, Feb, and Mar, and only a few days
suffered mild stress, nevertheless, most pregnant Hu sheep underwent
high and even severe stress in Jun, Jul, and Aug (Fig. [55]1c). The
rectal temperature and respiration rate of pregnant Hu sheep in Jun,
Jul, and Aug were higher than those in Jan, Feb, and Mar (Fig. [56]1d,
e).
Fig. 1. Establishment of the heat-stressed model in Hu sheep during
pregnancy.
[57]Fig. 1
[58]Open in a new tab
a Ambient temperature of pregnant Hu sheep shed. b Effective
temperature of pregnant Hu sheep. c THI of pregnant Hu sheep. d Rectal
temperature of pregnant Hu sheep (n = 10, ten Hu sheep in each group).
e Respiration rate of pregnant Hu sheep (n = 10, ten Hu sheep in each
group). The values were presented as mean ± SEM.
Body measurement traits of normal and IUGR lambs
The IUGR lambs exhibited significantly lower weight, height, chest
depth, chest width, chest circumference, and circumference of the
forearm bone compared to the normal lambs (P < 0.01, Fig. [59]2).
However, there was no significant difference in body length between the
two groups (P > 0.05, Fig. [60]2b). These findings suggest that heat
stress during pregnancy had a negative impact on the body measurement
traits of the IUGR lambs.
Fig. 2. Body measurement traits of the normal and IUGR lambs (n = 6, six
lambs in each group).
[61]Fig. 2
[62]Open in a new tab
a Weight of the normal and IUGR lambs. b Body length of the normal and
IUGR lambs. c Height of the normal and IUGR lambs. d Chest depth of the
normal and IUGR lambs. e Chest width of the normal and IUGR lambs. f
Chest circumference of the normal and IUGR lambs. g Circumference of
forearm bone in the normal and IUGR lambs. The values were presented as
mean ± SEM.
Isolation and identification of exosomes from umbilical plasma
Transmission electron microscopy analysis revealed the presence of a
significant number of “cup and plate” exosomes in the umbilical plasma
of both IUGR and normal lambs (Fig. [63]3a). The mean size of the
exosomes was 139.95 nm in the CON group and 130.95 nm in the HS-IUGR
group, as shown in Supplementary Table [64]1. Nanoparticle tracking
analysis indicated that exosomes with a diameter of 138.2 nm accounted
for 99.3% of the CON group, while exosomes with a diameter of 126.9 nm
accounted for 98.9% of the HS-IUGR group (Fig. [65]3b). The results of
our study showed that the average particle size of exosomes in IUGR
lambs was significantly decreased (P = 0.001, Fig. [66]3c). In
addition, our western blot analysis revealed that the extracellular
vesicles expressed CD81 protein, which is a surface marker protein of
exosomes. Calnexin is an endoplasmic reticulum-related protein, which
accelerates protein folding and assembly. Compared to 293T cells, there
was no Calnexin protein in exosomes. This finding indicated that the
separated vesicles were indeed exosomes and free of somatic cell
contamination derived from umbilical plasma in both IUGR and normal
lambs, as demonstrated in Fig. [67]3d. The Micro BCA protein assay was
utilized to compare the protein levels of exosomes that were isolated
from umbilical plasma. The results indicated that there was no
significant difference in the concentration of exosomal protein between
the CON and HS-IUGR groups (P > 0.05, Supplementary Table [68]1).
Fig. 3. Isolation and identification of exosomes from umbilical plasma in
normal and IUGR lambs (n = 3, three exosome samples in each group).
[69]Fig. 3
[70]Open in a new tab
a Representative images of exosomes separated from umbilical plasma by
transmission electron microscopy (TEM) (scale bar, 200 μm, and 200 nm).
b Representative photos of exosomes from normal and IUGR lambs by
nanoparticle tracking analysis, demonstrating the diameter and particle
distribution of separated exosomes. c Analysis of exosomal average
particle size. d Representative images of exosomal markers CD81 by
western blot. The values were presented as mean ± SEM.
Small RNA sequencing and small RNA distribution
To determine the concentration of exosomal RNA, the Quantus Fluorometer
was used, and it was found that the total RNA concentration of exosomes
in umbilical plasma-derived exosomes from IUGR and normal lambs ranged
between 0.16 and 0.60 ng/μl. Figure [71]4a displays the analysis
process for the small RNA sequencing. The sequencing base quality met
Q30, which was suitable for subsequent analysis (Fig. [72]4b). The
length of small RNA ranged from 17 to 147 bp (Fig. [73]4c). The
original reads had a total length of 93,610,573, with an average
reading length of 17,833,856.3 for the CON group and 13,369,668 for the
HS-IUGR group (Supplementary Table [74]2). After quality filtering, a
large proportion of raw reads remained, with clean_GC accounting for
over 50% of raw_GC (Supplementary Table [75]2). This suggested that the
sequencing quality of small RNA from exosomes derived from umbilical
plasma in both the CON and HS-IUGR groups was high. To remove ncRNA
from the clean reads, including rRNA, snoRNA, sRNA, and lncRNA, which
were compared to the Rfam library and discarded any matches. Then the
remaining reads were mapped to the sheep genome, and the mapped rate
was obtained (Supplementary Table [76]3). The comparison of Rfam
revealed that rRNA made up the largest portion of ncRNA, as shown in
Fig. [77]4d. Furthermore, the sequencing data indicated that the
majority of miRNAs in exosomes from umbilical plasma were 21–22 nt in
length, as demonstrated in Fig. [78]4e.
Fig. 4. Small RNA sequencing results of exosomes derived from umbilical
plasma in normal and IUGR lambs (n = 3, three exosome samples in each group).
[79]Fig. 4
[80]Open in a new tab
a Analysis process of small RNA sequencing. b Quality distribution of
small RNA sequencing. c Distribution of small RNA length in exosomes. d
Analysis of the clean reads by Rfam. e Distribution of expressed miRNA
length.
Differential expression analysis of exosomal miRNAs
In total, 71 miRNAs were identified in the exosomes of umbilical plasma
from both the CON and HS-IUGR groups. In addition, 62 and 63 miRNAs
were expressed in exosomes from the umbilical plasma of normal and IUGR
Hu lambs, respectively. Nevertheless, only two miRNAs showed
significant expression when compared to sheep precursors in miRBase,
based on the screened standard of |log2 Fold Change | ≥ 1 (P < 0.05,
Fig. [81]5a). There were 42 common miRNAs between the CON and HS-IUGR
groups, with eight unique miRNAs in CON and nine in HS-IUGR
(Fig. [82]5b). Notably, oar-miR-411a-5p was significantly downregulated
and oar-miR-200c was significantly upregulated in HS-IUGR compared to
the CON (P < 0.05, Fig. [83]5c). In Fig. [84]5d, the log2 Fold Change
of differentially expressed miRNAs was presented.
Fig. 5. Differentially expressed miRNAs of exosomes originated from umbilical
plasma in CON and HS-IUGR groups (n = 3, three exosome samples in each
group).
[85]Fig. 5
[86]Open in a new tab
a Volcanic map of miRNAs. The same miRNA may derive from different
predecessors, so some points coincide. b Venn diagram shows the common
and unique miRNAs of CON and HS-IUGR groups. c Heatmap of
differentially expressed miRNAs. d Histogram of differential miRNA
expression. The Y axis shows mature miRNA/precursor miRNA.
Target genes prediction of differentially expressed miRNAs
To predict the target genes of oar-miR-200c and oar-miR-411a-5p, which
were differentially expressed miRNAs found in exosomes derived from
umbilical plasma, MiRanda was used. The analysis revealed 1700 and 1029
unique target genes for oar-miR-200c and oar-miR-411a-5p, respectively.
Interestingly, there were 291 common genes between the two groups, as
shown in Supplementary Fig. [87]1. The common genes of oar-miR-200c and
oar-miR-411a-5p were illustrated in Supplementary Fig. [88]1.
GO and KEGG enrichment analysis of differentially expressed miRNAs
GO and KEGG enrichment analyses were conducted to determine the roles
of target genes in various biological processes (Fig. [89]6). The
findings revealed that 291 target genes were commonly enriched in 68 GO
terms, including 18 terms related to biological processes, 24 terms
related to cellular components, and 26 terms related to molecular
functions. Notably, these target genes were significantly enriched in
several key pathways, such as the Wnt signaling pathway, cell adhesion,
and cytoskeleton organization (P < 0.05, Fig. [90]6a). Furthermore, the
target genes exhibited significant enrichment in 35 KEGG pathways, such
as the cAMP, TGF-beta, calcium, and Rap1 signaling pathways (P < 0.05,
Fig. [91]6b).
Fig. 6. Enrichment analysis of common target genes of oar-miR-200c and
oar-miR-411a-5p.
[92]Fig. 6
[93]Open in a new tab
a GO enrichment analysis. b KEGG enrichment analysis.
Validation of differentially expressed miRNAs
To confirm the precision of small RNA-seq and investigate the role of
miRNAs in the development of Hu lambs, RT-qPCR was performed to assess
the expression levels of differentially expressed miRNAs, specifically
oar-miR-411a-5p and oar-miR-200c. The findings of this study
demonstrated that the expression of oar-miR-411a-5p was significantly
reduced, while oar-miR-200c was significantly increased in HS-IUGR
compared to the CON (P < 0.05, Fig. [94]7b). Furthermore, the
expression levels of the differentially expressed miRNAs were
consistent with the small RNA-seq results, which suggested that the
sequencing accuracy was high (Fig. [95]7a).
Fig. 7. Expression analysis of significantly expressed miRNAs in exosomes
derived from umbilical plasma of normal and IUGR lambs (n = 3, three exosome
samples in each group).
[96]Fig. 7
[97]Open in a new tab
a Expression of oar-miR-411a-5p and oar-miR-200c in exosomes detected
by small RNA-seq. b Relative expression level of oar-miR-411a-5p and
oar-miR-200c in exosomes determined by RT-qPCR. The values were
presented as mean ± SEM.
Discussion
Global warming, an increasingly prevalent climate phenomenon, has a
detrimental effect on the health and productivity of both humans and
animals due to continuous hyperthermia^[98]20. To gain a better
understanding of this impact, we have conducted various studies on heat
stress and exosomes in vivo and in vitro^[99]19,[100]21,[101]22. Hu
sheep are a locally protected breed of livestock and poultry in China,
originating from the Tai Lake basin in both Jiangsu and Zhejiang
provinces. They are known for their long estrus cycle and high litter
size^[102]23. However, heat stress can negatively impact the
reproduction of Hu sheep and may lead to the occurrence of IUGR.
Therefore, we chose Hu sheep as our experimental animals. To evaluate
the levels of heat stress, we used THI, a common index that utilizes
climate parameters, which is used to measure the level of heat stress
in animals. When THI is less than 72, there is no stress. If THI falls
between 72 and 79, there is mild stress. High stress occurs when THI
ranges from 80 to 90, and severe stress is indicated when THI exceeds
90^[103]24. In this study, pregnant Hu sheep were found to be in a
no-heat-stressed state for the majority of the time, with only a few
days in January, February, and March showing mild stress. However,
during June, July, and August, pregnant Hu sheep experienced high and
even severe stress. To evaluate the degree of heat stress, rectal
temperature, and respiration rate are considered key
indicators^[104]25. The study found that heat-stressed Hu sheep had
higher rectal temperatures and respiration rates compared to
non-heat-stressed Hu sheep. These results suggest that a model for
heat-stressed Hu sheep during mid-late gestation has been established.
To induce IUGR animal models, researchers altered the ambient
temperature. For example, Limesand et al. (2006) increased the ambient
temperature of ewes during late pregnancy to obtain IUGR lambs^[105]9.
This study found that exposing Hu sheep to heat stress during mid-late
gestation resulted in the birth of IUGR lambs. The improper prenatal
environment caused cellular stress and death in the placenta, which in
turn hindered fetal growth^[106]26. The dysfunction of the placenta led
to IUGR, as there were discrepancies between the metabolic needs of the
fetus and the placental supply^[107]27. The weight, height, chest
depth, chest width, chest circumference, and circumference of the
forearm bone were significantly decreased in IUGR lambs compared to
normal lambs (P < 0.01). This reduction in growth is due to the
placenta’s inability to supply sufficient nutrients and oxygen to the
fetus^[108]28. In addition, IUGR has long-lasting effects on organ
function^[109]26. There were other differences between the normal and
IUGR lambs, which might be resulted from genetic factors.
Several studies have utilized sheep as a model for in vitro experiments
related to exosomes. These studies have revealed a correlation between
exosomes and the development of sheep embryos. Specifically,
researchers have observed differences in the expression of mRNA, miRNA,
and proteome in the extracellular vesicles of uterine fluid between
pregnant sheep at 14 days and non-pregnant sheep. Furthermore, it has
been discovered that exosomes secreted by the ovine uterus can be
absorbed by both embryos and endometrium^[110]29,[111]30. In sheep, the
acetylcholinesterase content in serum, which represents the vitality of
exosomes, remained stable during mid-gestation. However, it decreased
in pregnant sheep on gestational day 133^[112]31. This suggests that
the content of exosomes in serum changes during pregnancy. Furthermore,
the miRNAs of exosomes from serum, umbilical serum, and placentomes
were sequenced, and it was discovered that miRNAs found in serum
exosomes can target cell growth, proliferation, and organ development
pathways^[113]19. In addition, miRNAs found in exosomes from umbilical
serum and placentomes were found to target cellular, biological, and
embryonic development signaling pathways^[114]19. These findings
demonstrate that exosomes play a crucial role in ovine pregnancy by
facilitating direct and dynamic communication between the embryo and
the maternal environment. This study found that IUGR lambs had a
reduced average particle size and the number of exosomes, suggesting
that maternal heat stress inhibited exosomal biogenesis and function in
the umbilical cord blood of IUGR lambs, ultimately leading to the
occurrence of IUGR.
Exosomes play a crucial role in modulating reproduction, pregnancy, and
embryo development in both humans and animals^[115]16. Exosomes are
small vesicles that contain a variety of molecules, including mRNAs,
proteins, and non-coding RNAs such as miRNAs, lncRNAs, and
circRNAs^[116]32. These vesicles play a crucial role in mediating
biological processes by facilitating cell-to-cell communication as
conduction molecules^[117]33–[118]36. Recent studies have shown that
miRNAs are distributed in various body fluids, including blood, saliva,
urine, sperm, and milk, as revealed by small RNA sequencing^[119]37.
Exosomal miRNAs have been found to regulate the development of
mammalian embryos, with miR-766-3p, miR-663b, and miR-132-3p from
exosomes having a significant impact on embryo development^[120]38.
Exosomes derived from follicular fluid contain miR-134, miR-323-3p, and
miR-410, which play a role in embryo development^[121]39,[122]40. When
the scrotum is exposed to heat stress, the miRNA content of small
extracellular vesicles decreases, leading to an impact on the function
of the testis and epididymis, and miR-126-5p may be transferred between
small extracellular vesicles and sperm, which is associated with
spermatogenesis and maturation of bovine sperm^[123]41. Up to this
point, no studies have reported on the expression of exosomal miRNAs
derived from umbilical plasma between IUGR and normal lambs. We
hypothesize that heat stress alters the composition of exosomal miRNAs
from umbilical plasma. In this study, we aimed to demonstrate the
differential expression of exosomal miRNAs from umbilical plasma
between IUGR and normal lambs. We identified 71 miRNAs including two
differentially expressed miRNAs of exosomes derived from umbilical
plasma in both IUGR and normal lambs. There were 13 significantly
expressed miRNAs of exosomes in the umbilical arterial serum compared
to the umbilical venous serum of lambs^[124]19. 116 and 226 miRNAs of
umbilical cord blood were differentially expressed in IUGR piglets and
IUGR fetuses^[125]42. The differentially expressed miRNAs were less
than the previous studies, the discrepancy may be due to the difference
in species. Specifically, oar-miR-411a-5p was significantly
downregulated and miR-200c was significantly upregulated in HS-IUGR
(P < 0.05). This difference in expressed miRNAs is in accordance with
the cellular function^[126]43, indicating a potential role in the
pathogenesis of HS-IUGR.
The GO enrichment analysis elucidated that the target genes were
enriched in a total of 68 GO terms. Of these, 18 were related to
biological processes, 24 were related to cellular components, and 26
were related to molecular functions. In addition, the KEGG pathway
enrichment analysis showed that the target genes were enriched in
several pathways, including the Wnt, cAMP, TGF-beta, calcium, and Rap1
signaling pathways. The signaling pathways identified in previous
studies have been shown to affect the development of skeletal
muscle^[127]44–[128]48. Based on this knowledge, we hypothesized that
heat stress experienced during pregnancy may have altered the miRNAs
present in exosomes found in umbilical plasma. This alteration could
then have led to the modulation of target genes, ultimately disrupting
the signaling pathways and impairing the development of skeletal muscle
in IUGR lambs. However, this hypothesis was not effectively tested in
this study.
The differences in the umbilical cord blood are a consequence of IUGR.
During pregnancy, miRNAs derived from umbilical cord blood make a
difference in fetal development^[129]42. In a study conducted by Luo et
al.^[130]42, it was found that there were differences in the exosomal
miRNAs of umbilical cord blood between IUGR and normal piglets, as
confirmed by transcriptome sequencing. These findings are consistent
with our results^[131]42. Therefore, further exploration of exosomes
derived from umbilical cord blood is necessary to shed light on the
relationship between miRNA expression and fetal development under heat
stress.
In conclusion, the heat stress experienced by Hu sheep during mid-late
gestation resulted in the occurrence of IUGR. Exosomes derived from
umbilical plasma were isolated and identified from both IUGR and normal
lambs, and a small RNA sequencing was conducted to detect the
expression of exosomal miRNAs. Heat stress has been found to alter the
expression of exosomes derived from umbilical plasma. Specifically, two
miRNAs, oar-miR-411a-5p and oar-miR-200c, have been identified as
potentially having a significant impact on fetal development in
heat-stressed Hu sheep through various signaling pathways. This study
provides new insights into the potential role of exosomal miRNAs in the
pathogenesis of IUGR and the effects of heat stress on fetal
development.
Methods
Inclusion and ethics
All experimental procedures were approved by the Animal Care and Use
Committee of Nanjing Agricultural University and were strictly
conducted according to the animal experiment guidelines (Approval ID:
SYXK 2022-0031; Approval Date: 2022-6-10). We have complied with all
relevant ethical regulations for animal testing.
Determination of environmental indicators and thermal parameters
The temperature in the sheep shed was measured daily during the months
of January, February, March, June, July, and August using a dry and
humidity thermometer. Both dry bulb temperature (Td) and wet bulb
temperature (Tw) were recorded. The effective temperature (ET) and
temperature-humidity index (THI) were calculated using the
formula^[132]24:
[MATH: THI=0.72×
(Td+Tw)+40.6<
/mn>,ET=0.35Td+0.65Tw :MATH]
In addition, rectal temperature and respiration rate were measured
using the method^[133]49. To obtain the rectal temperature of the
pregnant Hu sheep, an animal thermometer was inserted approximately
three to five cm into the rectum. After waiting for 5 min, the
temperature value was read. Additionally, the respiratory rate,
indicated by the fluctuation frequency of the lumbar fossa, was
observed during a fixed time while the Hu sheep were at rest. The
rectal temperature and respiration rate were measured on the 1st, 4th,
7th, 10th, 13th, 16th, 19th, 22nd, 25th, 28th, and 31st (30th) in Jan,
Feb, Mar, Jun, Jul, and Aug.
Sample collection
The compatriots or half-compatriots grown-up and healthy female Hu ewes
were selected with the same parity and age stage using synchronous
estrus and artificial insemination techniques so that the Hu fetuses
were born in March and August of the same year, respectively. The
pregnant ewes were fed under the same management conditions at Taicang
sheep farm, Jiangsu province, China. The pregnant ewes were fed the
silage, and the nutritional composition of the silage is shown in
Supplementary Table [134]4. The pregnant Hu sheep had ad libitum access
to feed and water throughout the gestation period. Each pregnant Hu
sheep was raised in a single pen. The body condition of the pregnant
ewes was observed every day. The weight of the lambs was measured upon
completion of the pregnancy, and IUGR lambs were selected based on a
standard that required their weight to be lower than two standard
deviations of the average weight for their age. The umbilical venous
blood samples were collected immediately after the Hu lambs were
delivered from two groups: six normal male lambs (CON group) born in
March, and six IUGR male lambs (HS-IUGR group) born in August. EDTA2K
blood collection tubes were used to collect the blood samples, which
were then mixed with an anticoagulant by inverting the tubes five
times. After collection, the umbilical venous blood was centrifuged at
1900×g for 10 min at 4 °C by a small refrigerated centrifuge (Beckman,
Microfuge 20 R), followed by a second centrifugation at 3000 ×g for
15 min at 4 °C. The separated plasma from each sample was collected and
stored in a 1.5-mL centrifugal tube at −80 °C for future use.
Body measurement traits of the Hu lambs
Various physical characteristics of both normal and IUGR lambs were
measured, including body length, height, chest depth, chest width,
chest circumference, and forearm bone circumference.
Extraction and identification of exosomes
To extract exosomes from umbilical plasma, an ultracentrifuge (Hitachi,
CP100MX) was utilized. The frozen plasma sample (4 mL) was thawed at
37 °C, and the resulting solution was transferred to a new tube and
centrifuged at 2000×g for 30 min at 4 °C. The supernatant was carefully
transferred to a new tube and centrifuged at 10,000×g for 45 min at
4 °C. The resulting supernatant was then filtered using a 0.45-μm
filter membrane, and the filtrate was collected. Next, the filtrate was
transferred to a new centrifuge tube and centrifuged with an overspeed
rotor at 100,000× g for 70 min at 4 °C. The pellet was resuspended in
10 mL of precooled 1× PBS. The overspeed rotor was utilized for another
round of centrifugation at 100,000× g for 70 min at 4 °C. Exosomes were
resuspended with 300 μL of precooled 1× PBS before being stored at
−80 °C ultra-low-temperature freezer (Thermo, 905) for downstream
applications.
RNA isolation
Exosomes (100 μL) were mixed with RNA lysate (700 μL). Following this,
chloroform (140 μL) was added and vortexed for 15 s. The tubes were
then incubated at room temperature for 3 min before being centrifuged
at 12,000× g for 15 min at 4 °C. The upper aqueous phase was
transferred to a new EP tube and mixed with 525 μL of absolute ethyl
alcohol in a 1.5-ml tube using a pipette (Eppendorf, Research Plus).
Next, a mixed solution containing all precipitates was transferred to
the RNeasy adsorption column and centrifuged at 8000× g for 15 s. The
filtrate was then discarded, and this step was repeated for the
remaining mixture. Buffer RWT (700 μL) and Buffer RPE (500 μL) were
used to wash the adsorption column, which was centrifuged at 8000× g
for 15 s, and the filtrate was discarded, respectively. Buffer RPE
(500 μL) was used to wash the adsorption column, centrifugation at
8000× g for 2 min. Then the adsorption column was transferred to a new
2-mL centrifuge tube, centrifuged at 12,000× g for 1 min to dry, and
the filtrate and collecting tube were discarded. Finally, the
adsorption column was transferred to a new 1.5-mL centrifuge tube.
Then, RNase-free water (30 μL) was added to the adsorption membrane,
and the mixture was centrifuged at 8000× g for 1 min to elute the RNA.
To determine the concentration of RNA, the extracted RNA (1 μL) was
stained and analyzed using a nucleic acid analyzer (Promega, Quantus
Fluorometer). The RNA was then stored at −80 °C.
Transmission electron microscopy
A 10-μL exosome sample was dripped onto a 200-mesh copper mesh (Beijing
Zhongjingkeyi Technology Co., Ltd., BZ11022a) and allowed to
precipitate for 1 min. Next, 1% uranium acetate (10 μL) was dripped
onto the copper mesh and allowed to precipitate for 1 min, with the
floating liquid again absorbed by the filter paper. The sample was left
to drip for several minutes before being observed under a transmission
electron microscope (Hitachi, HT-7700) at 100 kV to obtain images of
the exosomes.
Nanoparticle tracking analysis
The frozen exosome samples were thawed by immersing them in water at
25 °C and then placed on ice. After diluting the samples with 1× PBS,
we used a nanoparticle tracking analyzer (PARTICLE METRIX, ZetaVIEW) to
detect them. Each sample was imaged in six photos, including the field
of view at 1 μm, 100 nm, 200 nm, and 500 nm. Six samples were analyzed
and three duplicates were in each group.
Cell culture
The 293 T cell line preserved in our laboratory was cultured with DMEM
(Gibco, USA) containing 10% fetal bovine serum (FBS) (Gibco, USA) and
1% penicillin/streptomycin (Gibco, USA). The culture medium was changed
every two days. The cells were cultured in a 5% CO[2] incubator at
37 °C.
Protein extraction
The frozen exosomes were thawed at 37 °C to prepare the exosome
samples, and 5× RIPA lysis buffer was added promptly. The mixture was
then placed on ice for 30 min. The concentration of the standard sample
was determined using the BCA kit, with 5 μL of the sample added to the
BCA mixture. After incubation at 37 °C for 30 min, the absorbance value
was measured at OD 562 nm using a microplate reader (Thermo, Varioskan
LUX). The concentration of the exosome samples was calculated according
to the standard curve.
Western blot
To create the SDS-PAGE gel, a 12% concentration was used based on the
protein size of the sample. The protein samples mixed with the SDS
sample loading buffer (5×) (Solarbio, Beijing, China) were boiled at
95 °C in a metal bath for 5 min before being added to the
electrophoresis gel along with the marker (Thermo Pierce, 26616). After
electrophoresis, the gel was fixed and a suitable PVDF membrane (absin,
Shanghai, China) was cut and activated in methanol for 20 s. The
exosome protein was then transferred to the PVDF membrane. After the
transfer, the PVDF membrane was blocked with 5% skimmed milk diluted in
1
[MATH: × :MATH]
TBST (Solarbio, Beijing, China) for one hour. Next, the membrane was
cut and incubated overnight at 4 °C in a dilute primary antibody
calnexin (Proteintech, 10427-2-AP) with a dilution ratio of 1:5000 and
CD81 (SAB, 41779) with a dilution ratio of 1:1000. In all, 1× TBST was
used to wash the membrane three times, each time for 10 min. Finally,
the membrane was incubated at room temperature for 1 h in a dilute Goat
anti-Rabbit IgG Peroxidase Conjugated antibody (Merck Millipore,
AP132P) with a dilution ratio of 1:5000. In total, 1× TBST was used to
wash the membrane three times, each time for 10 min. Next, an equal
volume of mixed ECL A/B solution (Biosharp, Hefei, China) was added to
the membrane protected from light for 5 min. Finally, the
ultra-sensitive chemiluminescence gel imaging system (CLINX, ChemiScope
3000mini) was used to expose the image.
cDNA library purification
To construct the cDNA library, we connected the total RNA of exosomes
with a 3’ adapter and a 5’ adapter and performed reverse transcription
polymerase chain reaction (RT-PCR). Next, QMN Beads (143 μL) (Qiagen,
331505) were added to the reverse transcript product, mixed by
vortexing for 3 s, and incubated for 5 min after brief centrifugation.
The solution was then placed on a magnetic rack, and the supernatant
was removed after the magnetic beads had completely adsorbed it. In
all, 80% ethanol (200 μL) was added to the mixture. The supernatant was
then removed, and this process was repeated once. The remaining liquid
was absorbed and dried for 10 min. Next, nuclease-free water (17 μL)
was added to cover the magnetic beads. The mixture was then incubated
for 2 min after being thoroughly mixed. Once the magnetic beads had
completely adsorbed the supernatant, the resulting liquid (15 μL) was
transferred to a new centrifuge tube. The cDNA was purified and stored
at −20 °C.
Small RNA sequencing
To determine the expression of miRNAs in umbilical plasma exosomes of
IUGR and normal lambs, small RNA sequencing of the exosomes was
conducted using the PE150 sequence. We analyzed six samples of
exosomes, which included C01, C02, and C03 of the CON group, and H01,
H02, and H03 of the HS-IUGR group. The sequencing library’s quality was
assessed using fastqc. To improve the quality, fastp was utilized to
perform N-base excision, q20 filtration, and adapter excision at both
ends of the sequence. The Rfam library was compared to remove ncRNAs,
such as rRNA, tRNA, and snoRNA, using the bowtie short sequence
alignment tool. Small RNAs were quantitatively analyzed using miRDeep2.
The normalized miRNAs were then compared between the CON and HS-IUGR
groups. The differentially expressed miRNAs were analyzed by DESeq2 to
show the expression level between the two groups.
Prediction of target genes and GO and KEGG enrichment analysis
miRNAs bind to target genes by complementary pairing with the 3’UTR
sites. To predict the target genes of differentially expressed miRNAs
in sheep mRNA, the 3’UTR sequence was analyzed using MiRanda. The
selection of predicted target genes was based on a seed length greater
than 8 and an energetic score less than 10^−ΔΔG. We analyzed the
predicted target genes, focusing on functional enrichment through Gene
Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG)
enrichment analysis. GO is an internationally standardized system for
classifying gene function, which offers a comprehensive vocabulary for
describing the attributes of genes and gene products in organisms. This
system is dynamic and regularly updated to ensure accuracy and
relevance. GO is comprised of three ontologies that describe the
molecular function (MF), cell component (CC), and biological process
(BP). Each term in GO corresponds to an attribute, and the term is the
basic unit of GO. GO function analysis provides annotation on the GO
function classification of genes and significant enrichment analysis of
genes. To map each term, genes were transferred to the GO database
([135]http://www.geneontology.org/), and the number of genes associated
with each term was obtained. A hypergeometric test was conducted to
detect enriched GO terms in the genes relative to the sheep genome. The
Benjamin–Hochberg method (BH) was utilized to calculate the adjusted P
value (Padj), with Padj Δ.05 serving as the threshold for significant
enrichment.
To gain a deeper understanding of the biological function of the genes,
pathway analysis was conducted. The KEGG database is widely recognized
as the main public resource for pathway information and was therefore
used as the unit for pathway enrichment analysis. For more information
on the KEGG database, please visit [136]http://www.genome.jp/KEGG. A
hypergeometric test was utilized to identify pathways that exhibited
enrichment in genes when compared to the sheep genome. This pathway
enrichment analysis allowed for the identification of the most crucial
biochemical metabolism and signal transduction pathways that were
involved in the genes. The adjusted P value (Padj) was calculated using
the Benjamin–Hochberg method (BH).
Real-time quantitative PCR (RT-qPCR)
To ensure the precision of small RNA sequencing, RT-qPCR was conducted
to compare the expression levels of exosomal miRNAs that were
differentially expressed in the CON and HS-IUGR groups. The miRNA
primer sequences were synthesized by Beijing Tsingke Biotech Co., Ltd.
(Beijing, China). MiRNA 1st Strand cDNA Synthesis Kit (by stem-loop)
(Vazyme, MR101) was used to reverse transcribe the RNA of exosomes and
then determined the relative expression level of the differentially
expressed miRNAs using RT-qPCR^[137]50. The expression levels of
differentially expressed miRNAs were compared using the 2^−ΔΔCt method,
with U6 serving as the reference gene. The primer sequences used in the
analysis can be found in Supplementary Table [138]5.
Statistics and reproducibility
The data were presented as Mean ± SEM and analyzed using SPSS 23.0
(SPSS Inc., Chicago, IL, USA). One-way analysis of variance (ANOVA) was
conducted to compare the expression between the CON and HS-IUGR groups.
The figures were generated using GraphPad Prism 9 (La Jolla, CA, USA).
A level of P < 0.05 was considered statistically significant.
Reporting summary
Further information on research design is available in the [139]Nature
Portfolio Reporting Summary linked to this article.
Supplementary information
[140]Peer Review File^ (982.5KB, pdf)
[141]Supplementary Information^ (2.2MB, pdf)
[142]Description of Supplementary Materials^ (14.5KB, docx)
[143]Supplementary Data 1^ (67.6KB, xlsx)
[144]Reporting Summary^ (731KB, pdf)
Acknowledgements